{This set is designed in order to semiautomatically compute lengths of neurites of a given neuron (one neuron per slide). Before you can start, make an empty folder named 'analysis' inside the folder where you keep the images. Then, in the third line from the end of last macro, specify explicitly the route of 'analysis' folder typing the names of enclosed folders in order instead of Macintosh HD:*** given as an example. Another option is to change the whole line for SaveAs(''); . In this case you will be prompted every time for the name of results file. 1. Press F1 to prepare next neuron image for analysis. Select an image file to be opened, and press OK. 2. Ajust the contrast by selecting the LUT tool (sixth from the top in the right column of 'Tools' window) and moving the bar in the 'LUT' window. Neurites should appear as contiguous as possible while the background is not too noisy. 3. Use the pencil tool (third from the top in the left column of 'Tools' window) to connect the parts of each neurite. In order to see the picture in Grayscale, press with a little finger of your right hand. It is possible to use the pencil tool in Grayscale mode. When finished press again. 4. Use the auto-outline tool (tenth from the top in the left column of 'Tools' window) to highlight each subsequent neurite (click outside to the left of anywhere of a neurite), make sure that all neurite is outlined, then press to launch the computation. It is a good idea to use left hand only for this, because this is the last and most important step which should not be confused with previos ones (they usually can be undone while after wrong pressing you would have to go to step 1 and initialize image over). 5. If In order to save results of measurements, just press F5. The 'Results' window is then saved in 'analysis' folder under the name of data file. If you have any questions or suggestions please feel free to contact Victor Pikov at vipikov@vassar.edu} VAR Initialized: boolean; PictureName: string; Thresh:boolean; Thr:integer; macro 'Acquire [A]'; begin Acquire('CE/RT PlugIn 1.0') end; macro 'Initialize [F1]'; begin ShowResults; Open(''); ResetCounter; {Clear the old measurements} Initialized:=True; PictureName:=WindowTitle; SetOptions('Length'); WandAutoMeasure(false); SetScale(1.65,'5m'); SetThreshold(180); Thresh:=True; end; {Initialize} macro 'Grayscale/Threshold []'; var lower,upper:integer; begin if Thresh then begin GetThresholds(lower,upper); Thr:=lower; SetPalette('Grayscale'); Thresh:=False; exit; end; SetThreshold(Thr); Thresh:=True; end; {Gray/Thesh} macro 'Neurite arithmetics [ ]'; { Analyzes the current neurite selection and stores the results in the Area and Length columns. } begin If not Initialized then begin PutMessage('Use the INITIALIZE macro first.'); exit; end; {Make sure we're initialized first} Measure; rLength[rCount]:=rLength[rCount]/2; if (rArea[rCount]/rLength[rCount]>8) then begin rLength[rCount-1]:=rLength[rCount]+rLength[rCount-1]; SetCounter(rCount-1); end; UpdateResults; Clear; end; Macro 'Save the Work [F5]'; var i:integer; sum:real; begin SelectWindow('Results'); {Choose the results window} sum:=0; for i:=1 to rCount do sum:=sum+rLength[i]; SetCounter(rCount+1); rLength[rCount]:=sum; UpdateResults; SetExport('Measurements'); Export(concat('Macintosh HD:***:analysis:',PictureName)); {Save the measurements} DisposeAll; end; {Save the Work}